The renin-angiotensin (RA)system, essential to the regulation of blood pressure and aqueous electrolytes in vivo, plays a key role in various hypertensive diseases, congestive heart failure and edematous diseases.
Renin, produced mainly in the renal juxtaglomerular apparatus, acts on angiotensinogen, present in the blood, kidney and other organs, to produce angiotensin (Ang) I. Ang I possesses almost no bioactivity and, upon action of angiotensin-converting enzyme (ACE), is converted to a bioactive form known as Ang II. Ang II is thought to make a most significant contribution to the bioactivity of the RA system, though Ang III is also produced by the same system. The amino acid number of Ang III is lower by 1 than that of Ang II, and its bioactivity is similar to that of Ang II.
The most important action of Ang II is its cardiovascular action, its peripheral vasoconstrictive action being very potent and playing a major role in the maintenance of blood pressure. In addition to this action, Ang II has proven to be active on the adrenal zona glomerulosa to induce aldosterone production and on the adrenal medulla and sympathetic nerve ends to promote catecholamine secretion, vasopressin secretion and prostaglandin E2 and I2 production, and is involved in the glomerular filtering function and the renal uriniferous tubular sodium reabsorption mechanism. Since renin and Ang II are also produced in the brain, heart, vascular wall, adrenal and other non-kidney organs, the local action thereof as produced in these ectopic RA systems is drawing attention, as are the above physiological actions.
Since the RA system owes its bioactivity mainly to Ang II, as stated above, it has been believed that the above-mentioned diseases can be prevented or mitigated by suppressing Ang II production and hence the RA system. To inhibit the renin production, which is the starting material of the RA system, .beta.-blockers have long been used. However, since .beta.-blockers possess a broad range of action points, focus has recently been on the development of renin inhibitors, which are unsatisfactory in absorption via the digestive tract; no drug permitting practical application has been developed. Currently widely used RA system inhibitors are ACE inhibitors, exemplified by captopril, enalapril, delapril and alacepril. Although these drugs are already in practical application for their excellent effect, they have side effects, such as dry cough and diuresis, as a result of increase in bradykinin and prostaglandin levels, because they also suppress kininase II. Therefore the development of an Ang II receptor antagonist has been desired as a drug which suppresses only Ang II bioactivity.
Orally administrable non-peptide Ang II receptor antagonists have long been studied. For example, imidazole acetate series compounds have been studied for diuretic and hypotensive action since around 1976, and CV2961 and other compounds have been found to possess Ang II receptor antagonist activity [Y. Furukawa et al., U.S. Pat. No. 4,340,598 (1982); Y. Furukawa et al., U.S. Pat. No. 4,355,040 (1982)], as the result. Since then, there have been improvements in these compounds, resulting in the development of DuP 753 [A. T. Chiu et al., Journal of Pharmacology and Experimental Therapeutics, 252, 711 (1990)]. DuP 753 proved to act specifically on Ang II receptors in the vascular wall, adrenal cortex and other organs to suppress Ang II action only. It was also shown to have no effect on reactions of KCl, norepinephrine, isoproterenol, vasopressin, bradykinin, acetylcholine or 5-HT, or on ACE action, even at suppressive doses for a high concentration (10 .mu.M) of Ang II [P. C. Wong et al., J. Pharmacol. Exp. Ther., 252, 719 ( 1990)]. According to the recognition of such pharmacological utility of non-peptide antagonists for Ang II receptors, the studies of Ang II receptors has been increased. It is conjectured that there are at least two kinds of Ang II receptor, according to reactivity to antagonists: type 1 receptors, to which DuP 753 is antagonistic, and type 2 receptors, to which PD123177 is antagonistic [P.B.M.W.M. Timmermans et al., Trends in Pharmaceutical Science (TIPS), 12, 55 (1991)], of which type 1 receptors are thought to play a key role in known Ang H-dependent diseases.
Thus development of Ang II type 1 receptor antagonists has been brisk. To accurately assess the antagonist activity of a drug, it is necessary to use cells or cell membrane fraction having Ang II type 1 receptors only. However, the receptors used in these studies are based on membrane fractions derived from laboratory animals (e.g., bovines, rats), which contain various receptors other than the desired Ang II receptor. For this reason, there is a need for preparing cells which specifically express human Ang H type 1 receptors and the use of such cells or cell membrane fractions to accurately determine the bioactivity of the antagonist in a pure system.